CN104284901A - Side-chain protected oligopeptide fragment condensation using subtilisins in organic solvents - Google Patents

Abstract

Method for enzymatically synthesising an oligopeptide, comprising the coupling of an (optionally N-protected) protected oligopeptide ester with an (optionally C-protected) protected oligopeptide nucleophile in an organic solvent or an organic solvent mixture having a water content of 0.1 vol% or less, by a subtilisin in any possible form.

Description

The protected oligopeptide fragments condensation of side chain of the subtilisin with an organic solvent

The present invention relates to the method for enzyme' s catalysis oligopeptides.

Peptide class (especially oligopeptides) has many application, such as, as medicine, food or forage component, agrochemicals or cosmetic composition.

Known oligopeptides can in the solution or the chemosynthesis in solid phase by the flow process of height optimization.But, still having some limitations property in chemical peptide symthesis, especially large-scale chemical peptide symthesis.Such as, be greater than 10-15 amino acid whose peptide to be difficult to synthesize in solid phase, because they have the tendency of formation tertiary structure (by so-called " hydrophobic collapse "), it is very difficult that described tertiary structure makes peptide extend, to make to need reagent excessive in a large number and amino acid to build module.In addition, owing to there is the peptide of a large amount of similar-length, the purifying of finished product normally cost ineffective.Therefore; usually synthesized by the combining of solid phase synthesis of protected oligopeptide fragments more than 10 amino acid whose peptides; after the solid phase synthesis of described protected oligopeptide fragments, carry out chemical condensation in the solution, 20 amino acid whose peptides are prepared in such as 10+10 condensation.The major drawbacks of the oligopeptide fragments condensation be chemically protected is, except using the situation of C-terminal Gly or Pro residue, in the activation of C-terminal amino-acid residue, racemization occurs.Therefore, chemoprotectant oligopeptide fragments condensation strategy be restricted to use C-terminal activation Gly and Pro residue, or due to less desirable diastereomer formation and have to process very difficult purifying.By contrast, enzymatic oligopeptides coupling completely avoid racemization, and has some other advantage compared to chemical peptide symthesis.For industrial application, based on kinetics methodology chemo-enzymatic peptide class synthesis concept (namely using the carboxyl group of activation) be the most attracting (for example, see Sewald and H.D.Jakubke is in " peptide class: chemistry and biology ", 1 ^streprint, Wiley-VCH Verlag GmbH publishes, Wei Yin Haimu 2002).

Chemoenzymic peptide symthesis may need the enzymatic coupling of the not protected oligopeptide fragments of side chain, and described oligopeptide fragments is used chemical synthesis, fermentation method or the combination by chemistry and enzymatic coupling step to synthesize separately.Some are disclosed about the enzymatic condensation of the completely not protected oligopeptides of side chain under water surrounding (Kumaran et al.Protein Science, 2000,9,734; et al.Bioorg.Med.Chem.1998,6,891; Homandberg et al.Biochemistry, 1981,21,3387; Komoriya et al.Int.J.Pep.Prot.Res.1980,16,433).But the major drawbacks of this enzymatic oligopeptide fragments condensation under aqueous systems is, there is oligopeptide amides key and C-terminal ester hydrolysis simultaneously, cause low yield and many by products.In order to reduce the amount of the hydrolysis of expensive oligopeptides starting raw material and peptide class product, the excessive in a large number oligopeptides nucleophilic reagent (5-10 equivalent) of usual use increases condensation speed, and therefore reduce hydrolytic side reactions, see economically that this is a kind of very not attractive strategy.In order to reduce the amount of hydrolysis further, in low water reaction mixture, used organic cosolvent to implement the condensation of the completely not protected oligopeptide fragments of enzymatic, show higher product yield and less hydrolytic side reactions (Slomczynska et al.Biopolymers, 1992,32,1461; Xaus et al.Biotechnol.Tech.1992,6,69; Nishino et al.Tet.Lett.1992,33,3137; Cl á pes et al.Bioorg.Med.Chem.1995,3,245, Kolobanova et al.Russian J.of Bioorg.Chemistry2000,26,6,369).Because in these reports, need a large amount of water for the activity (water of 1-5 volume %) of enzyme, so hydrolytic side reactions is not still completely eliminated.In order to almost eliminate enzymically hydrolyse side reaction, almost water free reaction mixture (being less than the water of 1 volume %) can be used.But; under these conditions; considerably less enzyme is only had to be have active and stable (G.Carrea; S.Riva; Fundamentals of Biocatalysis in Neat Organic Solvents, Whiley, 2008); and in these organic solvents, the oligopeptides containing not protected side chain functionalities usually shows very little solvability or does not dissolve.Some reports disclose dipeptides and the enzyme' s catalysis of tripeptides in anhydrous organic solvent (such as Chen et al.J.Org.Chem.1992,57,6960), but do not implement the condensation of oligopeptide fragments.Although almost water free solvent almost eliminates hydrolytic side reactions, as a rule many enzymic activitys are lost, and therefore oligopeptides linked reaction trends towards slowly, and not exclusively.

As from liquid phase chemical peptide symthesis know, protected oligopeptides is soluble in the organic solvent of several non-water mixing due to its hydrophobicity.Therefore, protected oligopeptides is used to can be embodied in enzymatic oligopeptide fragments condensation in anhydrous organic solvent.But, can be expected that multiple side chain protected group of hydrophobic protection that spatially needs hinders enzyme identification.Such as, Yan et al.Tetrahedron, 2005,61,5933 report use protease subtilisin enzyme A, can not get condensation product completely with the protected amino acid of side chain.According to their observation, subtilopeptidase A gets rid of the amino-acid residue carrying the protecting group of large volume in its side chain functionalities; But when the protecting group of these large volumes is removed, amino-acid residue is easily accepted.

The people such as Gill (J.Am.Chem.Soc 1995,117,6175-6181) also illustrate a kind of method of the enzyme' s catalysis for oligopeptides.But the synthesis as described in the people such as Gill needs specific enzyme for each indivedual amino acid that adds to synthesize fragment, and needs other enzyme for the coupling of two fragments.Different enzymes must be used for the combination of two fragments, and the fact that another kind of enzyme is used for the condensation of two fragments makes the method not be suitable for plant-scale application.In addition, the fragment condensation step described by the people such as Gill needs two the side chain allyl-based protections removing one of them fragment, can realize coupling to make the use of V8 proteolytic enzyme.Therefore, the method described by the people such as Gill is not a kind of universal method of oligopeptides for the preparation of comprising 8 or more amino-acid residues.

Therefore, still exist and there is not or seldom exist the demand of the enzymatic means of the simple general-purpose of hydrolytic side reactions to for the synthesis of the peptide comprising at least 8 amino-acid residues.Such enzymatic method has been found at present unexpectedly.

Within the framework of the invention, by a kind of oligomeric peptide ester i) be defined as the oligomeric peptide ester comprising 4 or more amino-acid residues,

-comprise at least two amino-acid residues, the side chain functionalities that each amino-acid residue is protected with protected base, and comprise the C-terminal ester of activation, it is by C (=O)-O-CX ₂-C (=O) N-R ₁r ₂chemical formulation, wherein, each X represents hydrogen atom or alkyl or aryl independently, R ₁represent hydrogen atom or alkyl or aryl, R ₂represent hydrogen atom or alkyl or aryl or with the amino-acid residue of C-terminal Carboxylamide or carboxylic acid functional or peptide residue; described amino-acid residue or peptide residue are optionally in the side chain functionalities or protected in one or more side chain functionalities of peptide residue of amino-acid residue

-and wherein oligopeptides ester optionally comprises N-terminal protection.

To such as be used for the oligopeptides nucleophilic reagent ii of method of the present invention) be defined as the oligopeptides nucleophilic reagent comprising 4 or more amino-acid residues,

-comprise N-terminal amido, and at least two amino-acid residues, the side chain functionalities that each amino-acid residue is protected with protected base, and

-wherein, described oligopeptides nucleophilic reagent optionally comprises C-terminal protection.

Method of the present invention is a kind of method for enzyme' s catalysis oligopeptides, described method comprises the oligopeptides ester i of 4 as defined above or more amino-acid residues) with comprise the oligopeptides nucleophilic reagent ii comprising 4 or more amino-acid residues as defined above) coupling, describedly be coupled in the organic solvent or ORGANIC SOLVENT MIXTURES comprising and betide the water of 0.1 below volume % of total amount of liquid wherein significantly relative to linked reaction, carry out under the existence of subtilisin, wherein, the water by described enzyme r e lease in coupling reaction process is removed.

For purposes of the present invention, the per-cent of the water in solvent mixture is by the per-cent as the water measured by Karl Fischer volumetry carried out described in experimental section.

As above i) as described in oligopeptides ester also can be called as " acry radical donor " in this article, and oligopeptides nucleophilic reagent ii) be usually also referred to as " nucleophilic reagent ".

Adopt method of the present invention, having found may the protected oligopeptide fragments of enzymatic ground condensation.Especially, found unexpectedly, the oligopeptides acry radical donor (i.e. oligopeptides ester as above) with multiple side chain protected group is accepted as substrate by enzyme.Further, there is protected oligopeptides condensation, and there is no obvious side reaction.Adopt according to method of the present invention; the productive rate being greater than 80% can be obtained; this per-cent divided by the total mole number of remaining acry radical donor, required product and hydrolysate by the mole number of the acry radical donor by changing into required product, then is multiplied by 100% and calculates.Preferably, obtain the productive rate being greater than 90%, be most preferably greater than the productive rate of 95%.

Unexpectedly, the productive rate being greater than 80% can be obtained, even if use excessive considerably less or inexcessive coupling partner i) or ii) in one.This synthesize to be formed with the chemo-enzymatic peptide in water or in the few solution of water content and contrasts, and its needs nucleophilic reagent excessive very in a large number obtains the productive rate being greater than 80%, normally 5-10 equivalent.

The advantage of method of the present invention is, which provides difference and is to participate in the terminal amino acid residue of linked reaction, comprise the coupling of the multiple protected oligopeptides of non-protein amino acid (Non-proteinogenic amino acid).The C-terminal residue of C-terminal ester is used in regardless of which albumen or non-protein amino acid; all can not there is the racemization of this residue; but; when the fragment condensation be chemically protected; usual generation racemization; unless Gly or Pro is used to this site, therefore, method according to the present invention provides obviously freely more on fragment condensation strategy.

Further, the advantage of method of the present invention is, the hydrolysis degree of the ester moiety of protected oligopeptides C-terminal ester is low, is namely low in the typical time frame for completing coupling, at least in some experiments, does not observe the ester hydrolysis that can detect.For purposes of the present invention, " low " is restricted to the acry radical donor being less than 5%, in the method according to the invention, is normally hydrolyzed much smaller than the acry radical donor of 5%.The hydrolysis degree of the amido linkage of protected oligopeptide fragments, or the hydrolysis degree of peptide product prepared by enzymatic is also low, at least in some experiments, does not observe detectable hydrolysis.

For purposes of the present invention, " oligopeptides " refers to based on 2-200 amino acid whose peptide, especially based on 2-100 amino acid whose peptide, also especially based on 2-50 amino acid whose peptide, the peptide of the peptide of preferred any 2-200 amino acid whose straight chain, a preferred 2-100 or 2-50 amino acid whose straight chain, it will be used to according to method of the present invention.For purposes of the present invention, " peptide " refers to that this peptide is the product according to method of the present invention based on 8 or more amino acid whose any amino acid chains.

For purposes of the present invention; " protected oligopeptides " refers to any oligopeptides of the chain comprising at least 4 amino-acid residues; wherein at least two amino-acid residues have side chain functionalities, and wherein at least Liang Ge amino acid side chain functional group protected base protection respectively.Side chain functionalities is such as hydroxyl, carboxylic acid, primary amine or secondary amine (comprising indoles and guanidine radicals), mercaptan or carboxy and amide groups functional group.Be preferably greater than 60%, all side chain functionalities more preferably greater than 70% and more preferably greater than the existence of 80% are protected.

For purposes of the present invention, " not protected oligopeptides " refers to any oligopeptides having and be less than two protected amino-acid residues of its side chain functionalities.

In content of the present invention, " amino acid side chain " refers to any protein amino acid side chains or non-protein amino acid side chain.

Argine Monohydrochloride is the amino acid of being encoded by genetic code.Argine Monohydrochloride comprises: L-Ala (Ala), α-amino-isovaleric acid (Val), leucine (Leu), Isoleucine (Ile), Serine (Ser), Threonine (Thr), methionine(Met) (Met), halfcystine (Cys), l-asparagine (Asn), glutamine (Gln), tyrosine (Tyr), tryptophane (Trp), glycine (Gly), aspartic acid (ASP), L-glutamic acid (Glu), Histidine (His), Methionin (Lys), arginine (Arg), proline(Pro) (Pro) and phenylalanine (Phe).

Non-protein amino acid can be selected from D-amino acid, phenylglycocoll, DOPA (3,4-dihydroxyl-L-Phe), the fluoro-phenylalanine of beta-amino acids, 4-or C especially ^α-alkylating amino acid.

For purposes of the present invention, condensation refers to the formation of the new peptide bond between two oligopeptides.

Within the framework of the invention, amino-acid residue or peptide residue refer to amino acid or the peptide of the N-terminal amino having deducted this amino acid or peptide.

Be that the C-terminal carboxyl functional group provided with blocking group is provided at this term used " C-terminal protection ", usually protect in fact carboxyl in order to avoid be coupled to the amido of another molecule.C-terminal blocking group can be C-terminal ester, makes C-terminal carboxyl at least substantially protected and avoid being coupled on amine under used peptide symthesis condition with this.T-alkyl is conventional blocking group.C-terminal blocking group also can be C-terminal Carboxylamide.Uncle's Carboxylamide is conventional protecting group.C-terminal protecting group also can be hydrazides, carbamyl hydrazides or thioesters.C-terminal protection can be temporary transient or permanent, and the latter refers to that this protection part is the part of required finished product.

Term used herein " N-terminal protection " represents the N-terminal amido provided with blocking group, usually protects N-terminal amido at least in fact, in order to avoid participate in the coupling of C-terminal carboxyl and N-terminal amido.

Protected oligopeptides C-terminal ester i in the enzymatic condensation for protected oligopeptides) in Zhi – C (=O) O-CX ₂-C (=O) N-R ₁r ₂part is the ester of activation.The ester of activation is to provide the ester of the carboxyl ester that can participate in linked reaction.

At this, each X represents hydrogen atom or alkyl or aryl independently.In the method for the invention, when wherein each X is hydrogen (-O-CH ₂-C (=O) N-, is called Cam ester) time, obtain particularly preferred effect.

In described ester moiety, R ₁represent hydrogen atom or alkyl or aryl, R ₂represent hydrogen atom or alkyl or aryl; or with the amino-acid residue of C-terminal carboxylic acid amides or carboxylic acid functional or peptide residue, described amino-acid residue or peptide residue are optionally in the side chain functionalities or protected in one or more side chain functionalities of peptide residue of amino-acid residue.

In this article, use vocabulary " alkyl " or " aryl " part, use to give a definition: each alkyl can represent (substituted or unsubstituted) C1-C7 alkyl independently, preferably (substituted or unsubstituted) straight chain C 1-C6 alkyl, more preferably (substituted or unsubstituted) straight chain C 1-C3 alkyl, and most preferable.

Each aryl can represent (substituted or unsubstituted) C4-C13 aryl independently, preferably (substituted or unsubstituted) C4-C6 aryl, more preferably (substituted or unsubstituted) C6 aryl, and most preferably phenyl.Aryl optionally comprises one or more heteroatoms on its ring.Heteroatoms especially can be selected from the group of S, O and N.Substituting group on alkyl or aryl can be to stop oligopeptides ester i) and oligopeptides nucleophilic reagent ii) between there is any atom of linked reaction or the group of atom.Which substituting group those skilled in the art can easily check be applicable to maybe to be used for according to method of the present invention.

In the method for the invention, wherein R ₁and R ₂both represent hydrogen atom or R ₁represent hydrogen atom and R ₂represent the amino-acid residue with C-terminal Carboxylamide or carboxylic acid functional or peptide residue; described amino-acid residue or peptide residue optionally, in the side chain functionalities of amino-acid residue or time protected in one or more side chain functionalities of peptide residue, obtain particularly preferred effect.

In another embodiment, the C-terminal ester group of the activation of protected oligopeptides ester can be introduced in solid phase while racemization occurs with high yield and high purity and not.Use another advantage of ester to be, the C-terminal ester group of their activation can be introduced cheap and in industrial obtainable 2-chlorine trityl resin, wherein in described ester, and R ₁represent hydrogen atom and R ₂represent with the amino-acid residue of C-terminal carboxylic acid functional or peptide residue, described amino-acid residue or peptide residue are optionally in the side chain functionalities of amino-acid residue or protected in one or more side chain functionalities of peptide residue.

Protected oligopeptides ester i) C-terminal amino acid can be any amino acid (Argine Monohydrochloride or non-protein amino acid) in theory, and oligopeptides ester can be made up of Argine Monohydrochloride and/or non-protein amino acid.

Particularly (optionally N-terminal is protected) ester i of activating) can be represented by the compound of structural formula I.

At this, P represents hydrogen or N-terminal blocking group.Suitable N-terminal protecting group is that the N that those may be used for synthesizing (widow) peptide holds blocking group.These groups are as well known to those skilled in the art.Suitable N holds the example of blocking group to comprise carbamate or acyl group type protecting group; such as " Cbz " (carbobenzoxy-(Cbz)), " Boc " (tertbutyloxycarbonyl); " For " (formyl radical); " Fmoc " (9-fluorenylmethyloxycarbonyl), " PhAc " (phenylacetyl) and " Ac " (ethanoyl).For, PhAc and Ac group can use BPTI deformylase, PenG acyltransferase or acyltransferase enzyme to introduce and excise respectively.Chemical ablation method is known in the art.

In structural formula I, n represents the integer being at least 4.N especially can be at least 5, is at least 6, is at least 7, is at least 8, is at least 9, is at least 10.N especially can be 100 or following, 75 or following, 50 or following, 25 or following, 20 or following, 15 or following, such as 10 or following.

In formula I, each R ^awith each R ^brepresent hydrogen atom or organic group independently of one another, preferred amino acid side chain.Therefore, R ^awithout the need to all identical in all n Amino Acid Unit.Similarly, R ^bdo not need in all n Amino Acid Unit all identical.According in the oligopeptides ester of formula I, at least two each self-contained side chains with protecting group of amino-acid residue, namely for these amino-acid residues, R ^aand R ^bnot H, and respectively carry protected functional group, such as protected hydroxyl, carboxylic acid, primary amine or secondary amine (comprising such as indoles and guanidine radicals), sulfydryl or primary amide functional group, namely with protected side chain functionalities.Preferably, at R ^aand R ^bthe functional group of middle existence at least 50% should be protected by blocking group as known in the art altogether.Many different protecting groups are known, and can use in the method according to the invention.If use the Fmoc based on Solid phase peptide synthesis; so Side chain protective group can be selected from such as t-Bu (tertiary butyl), Boc, Trt (trityl), Mtt (4-methyltrityl), Acm (acetyl aminomethyl), Dnp (2; 4-dinitrophenyl), Pmc (2,2,5; 7; 8-pentamethyl-chroman-6-alkylsulfonyl) or Pbf (2,2,4; 6,7-pentamethyl-coumaran-5-alkylsulfonyl) group.If use the Boc based on Solid phase peptide synthesis, so Side chain protective group can be selected from and such as can be selected from Bzl (benzyl), Bz (benzoyl), 2Cl-Z (2-chlorobenzene methoxycarbonyl), cHex (cyclohexyl), Tos (tosyl group), Xan (xanthenyl), For or Mbzl (4-methoxy-benzyl) and 3-benzyloxymethyl group.

In a preferred embodiment, oligopeptides ester i) all side chain functionalities protected, except the side chain functionalities of the C-terminal amino-acid residue of oligopeptides acry radical donor, the C-terminal ester namely activated.The advantage of this embodiment is, than wherein oligopeptides acry radical donor i) the protected embodiment of side chain functionalities of C-terminal amino-acid residue obtain higher productive rate and/or shorter reaction times.

Ester i by with activation) (optionally, C-terminal is protected) protected oligopeptides nucleophilic reagent ii of coupling) can be any peptide based on Argine Monohydrochloride or non-protein amino acid in theory.

Especially, (optionally, C-terminal is protected) protected oligopeptides nucleophilic reagent ii) can represent with the compound of general formula I I:

Wherein, n, R ^aand R ^bas defined above.According to each self-contained side chain with protecting group of at least two amino-acid residues in the oligopeptides nucleophilic reagent of general formula I I, namely for each these amino-acid residue, R ^aand R ^bnot H, and there is protected functional group separately, such as protected hydroxyl, carboxylic acid, primary amine or secondary amine (comprising such as indoles and guanidine radicals), sulfydryl or primary amide functional group, namely with protected side chain functionalities.Preferably, at R ^aand R ^baltogether at least 50% should being protected by protecting group as known in the art of the functional group of middle appearance.

At this, Q represents OR part, amido, hydrazino, carbamyl-hydrazino or SR group.

When Q represents OR part, R can represent C-terminal protectiveness group, hydrogen atom or positively charged ion, such as univalent cation, such as three replacements or quaternary ammonium ion or alkali metal cation.As R during C-terminal protectiveness group, it can especially be (being optionally substituted) alkyl or (being optionally substituted) aryl.When R is C-terminal protectiveness group, R is preferably tertiary alkyl, although it also can be any other protectiveness group known in those skilled in the art in theory.This tertiary alkyl can be any protectiveness tertiary alkyl in theory.Preferably, this tertiary alkyl is selected from the group of the tertiary butyl (2-methyl-2-propyl), tert-pentyl (2-methyl-2-butyl) and tertiary hexyl (2,3-dimethyl-2-butyl).

When Q represents amido, this amido can use general formula NR ₃r ₄represent, wherein, R ₃and R ₄hydrogen atom, any (substituted or unsubstituted) alkyl or any (substituted or unsubstituted) aryl can be represented independently of one another.Especially, R ₃and R ₄in one can be hydrogen atom, another can be (substituted or unsubstituted) alkyl.Work as R ₃and R ₄particularly preferred result is obtained when being all hydrogen atom.

When Q represents hydrazino, this hydrazino can use general formula NR ₅-NR ₆r ₇represent, wherein R ₅, R ₆and R ₇hydrogen atom, any (substituted or unsubstituted) alkyl or any (substituted or unsubstituted) aryl can be represented independently of one another.Preferably, R ₅, R ₆and R ₇be hydrogen atom.

When Q represents carbamyl-hydrazino, this carbamyl-hydrazino can use general formula NR ₈-NR ₉c (O) NR ₁₀r ₁₁represent, wherein, R ₈, R ₉, R ₁₀and R ₁₁hydrogen atom, any (substituted or unsubstituted) alkyl or any (substituted or unsubstituted) aryl can be represented independently of one another.Preferably, R ₈, R ₉, R ₁₀and R ₁₁be hydrogen atom.

When Q represents SR part, R can represent C-terminal protectiveness group, hydrogen atom or positively charged ion, such as univalent cation, such as three replacements or quaternary ammonium ion or alkali metal cation.As R during C-terminal protectiveness group, it can be (being optionally substituted) alkyl or (being optionally substituted) aryl especially.When R is C-terminal protectiveness group, R is preferably tertiary alkyl, although it also can be any other protectiveness group known in those skilled in the art in theory.This tertiary alkyl can be any protectiveness tertiary alkyl in theory.Preferably, this tertiary alkyl is selected from the group of the tertiary butyl (2-methyl-2-propyl), tert-pentyl (2-methyl-2-butyl) and tertiary hexyl (2,3-dimethyl-2-butyl).

In the method for the invention, the coupling of protected oligopeptides ester and protected oligopeptides nucleophilic reagent carrys out catalysis by subtilisin (E.C.3.4.21.62).In theory, any subtilisin of this linked reaction of catalysis can be used.When mentioning the subtilisin from particular source, originate from the first organism, but in fact at the recombination bacillus subtilis proteolytic enzyme that (genetic modification) second is produced in organism, refer in particular to and be included as the enzyme from the first organism.

Preferably, the subtilisin used in the method for the invention is subtilopeptidase A.

Multiple subtilisin is known in the art, the document such as seen US 5316935 and quote.Such subtilisin can be used for according in method of the present invention.

Use subtilopeptidase A (being wherein used in the peptide ester i of C-terminal site with proline residue) in the method according to the invention) the usual linked reaction produced slowly.Therefore, in a preferred embodiment, method according to the present invention is implemented under restrictive condition, if namely reaction is when implementing under the existence of wild-type subtilopeptidase A, oligopeptides ester i) in C-terminal amino-acid residue be not proline residue.Same for other subtilisins, preferably use wherein C-terminal amino-acid residue not to be the oligopeptides ester i of proline residue).But, can be expected that, use the mutant of subtilopeptidase A can obtain acceptable coupling rate.

The example of the organism that can derive from the subtilisin used in the method for the invention comprises Trichoderma species, such as from Trichodermareesei; Head mold species, such as, from Rhizopus oryzae; Bacillus, such as, from Bacillus licheniformis, subtilis, bacillus amyloliquefaciens, Bacillus clausii, bacillus lentus, Alkaliphilic bacillus (bacillus alkalophilus), Alkaliphilic bacillus (bacillus halodurans); Aspergillus, such as, from aspergillus oryzae or aspergillus niger; Streptomyces, such as, from streptomyces caespitosus or streptomyces griseus; Mycocandida; Fungi; Humicola; Rhizoctonia; Addicted to Cellulomonas (Cytophagia); Mucor; And animal tissues, particularly from pancreas, as pig pancreas, ox pancreas or sheep pancreas.

It would be clear to one of ordinary skill in the art that the varient of (wild-type) subtilisin that also can use natural generation in the method according to the invention.The mutant of wild-type enzyme can such as by wild-type enzyme that modifying DNA is encoded, use well known to a person skilled in the art induced mutation technology (random mutation, site-directed mutagenesis, orthogenesis, gene shuffling etc.), go out to make DNA encoding and be from wild-type enzyme difference the enzyme that at least one amino acid is different, or shorter than wild-type to make it encode out, and by affecting the enzyme of the expression of DNA in suitable (host) cell of modifying thus.The mutant of enzyme may have the characteristic of improvement, such as about following one or more in: substrate spectrum, activity, stability, organic solvent tolerance, temperature curve, synthesis/hydrolysing rate and side reaction curve.

In a preferred method, subtilopeptidase A is used to catalyzed coupling reaction.Subtilopeptidase A can believe the subtilisin that (Novozymes) is purchased from Novi, and be found in condensation protected coupling partner with in the relatively short time with good productive rate produce needed for peptide prod in especially there is advantage.

it is the suitable source of subtilopeptidase A.This product can believe from Novi that (Bagsvaerd, Denmark) obtains. it is the cheap and industrial obtainable proteolytic enzyme mixt (comprising subtilopeptidase A as Major Enzymes composition) produced by Bacillus licheniformis (Bacillus licheniformis).

Commercially available enzyme such as may be provided in fluid form by provider, especially in form of an aqueous solutions.In this case, preferably enzyme first such as, is separated from causing the unwanted liquid of unwanted side reaction, excessive water or alcohol.This can suitably have been come by precipitation, is usually separated from liquid by solid subsequently, and/or dry.Alcohol, the such as trimethyl carbinol can be used precipitate.When using another kind of alcohol, should be noted that this alcohol adversely can not disturb linked reaction.

In preferred embodiments, enzyme is used with fixed form.At least in some embodiments, this can cause the productive rate of the increase of oligopeptides synthesized after the relatively short reaction times.Use Alcalase cross-linked enzyme aggregate (Alcalase-CLEA) or use the Alcalase be fixed on solid particulate, such as Alcalase-Imibond, Alcalase-Epobond, Alcalase-immozyme or Alcalase-Decalite, obtained especially good effect.The fixing of enzyme also can make enzyme easily restore after linked reaction, can be recovered or reuse in continuous print linked reaction to make it.

In inert organic solvents, implement enzymatic linked reaction is possible.Some examples of suitable solvent are such as N, dinethylformamide (DMF), N-methyl-pyrrolidon (NMP), N, N-N,N-DIMETHYLACETAMIDE (DMA), dimethyl sulfoxide (DMSO) (DMSO), acetonitrile, the hydrocarbon polymer of such as toluene, such as methylene dichloride, 1, the halohydrocarbon of 2-ethylene dichloride or chloroform, such as methyl tertiary butyl ether (MTBE), tetrahydrofuran (THF) (THF), 2-methyl-tetrahydro furans (Me-THF) or 1, the ether of 2-glycol dimethyl ether, or such as 2, 2, (halogenation) alcohol of 2-trifluoroethanol (TFE), or the mixture of these organic solvents.Preferably, enzymatic linked reaction can be carried out in the organic solvent containing MTBE, THF, Me-THF, 1,2-glycol dimethyl ether, methylene dichloride, 1,2-ethylene dichloride, TFE, DMF, NMP, DMA or DMSO or ORGANIC SOLVENT MIXTURES.Most preferably, enzymatic linked reaction can at mixture, the methylene dichloride containing MTBE, MTBE and DMF or NMP or DMA or DMSO, or carries out in the organic solvent of the mixture of methylene dichloride and DMF or NMP or DMA or DMSO or ORGANIC SOLVENT MIXTURES.

Enzymatic fragment condensation is implemented usually in the substantial absence of water.It will be understood by those skilled in the art that, depend on enzyme, a small amount of water can be need to be used for making enzyme can suitably play its catalytic activity.

Substantially anhydrously refer to that reaction medium is not moisture or containing the water of minimum, namely based on the entire volume of the liquid in reaction medium, content is the water of 0.1 below volume %.Reaction medium can be dispersed in second liquid mutually in or another liquid phase can be dispersed in reaction medium.When two-phase or multiphase system, concrete water-content based on linked reaction betide wherein mutually in the volume of liquid, or when wherein the linked reaction multiphase system at least mainly betided wherein exists.The higher limit of the expectation of water concentration depends on oligopeptides ester i) and oligopeptides agent ii) concentration, depend on specific enzyme, the solvent used, the character (size of such as peptide and aminoacid sequence) of peptide that will synthesize, required final transformation efficiency and desired speed of reaction.

In the method according to the invention, water concentration is 0.1 below volume %, preferably 0.05 below volume %, more preferably 0.01 below volume %.

Water concentration does not have lower limit at this, because may need the limit of detection of water lower than known analytical procedure of the minimum existed.This is also applicable for being used to detect according to the detectability of the karl Fischer volumetry of the water concentration of method of the present invention.In a preferred embodiment, can be removed continuously or off and on by the water of enzyme r e lease.In theory, remove water to have been come by mode well known in the art.What be highly suitable for removing water is evaporation, such as, use vacuum or distillation azeotropic to remove.Molecular sieve is used to obtain particularly preferred effect.But, importantly substantially keep the activity of required enzyme.

The molecular sieve adding various amounts in enzymatic linked reaction can make the change of water concentration lower than its detectability.The too low water concentration such as obtained by adding a large amount of molecular sieves can cause (part) enzyme deactivation gradually in coupling reaction process in some cases.Those skilled in the art easily can determine the best water concentration of certain linked reaction by the molecular sieve of different amount.In enzymatic coupling reaction process (part) enzyme deactivation situation in, the enzyme of (partly) inactivation can be come completely by the enzyme of this (partly) inactivation of stirring in aqueous or almost entirely reactivate by rehydration, such as.This reactivate can make it possible to reuse enzyme in continuous linked reaction.In some cases, especially use the enzyme of freeze-drying, before enzymatic coupling, enzyme require is hydrated, to obtain enough catalytic activitys.When on-fixed enzyme, such hydration can be implemented by stirring then precipitation in aqueous, such as, use easily mixed water-soluble organic solvent, the such as trimethyl carbinol.When immobilized enzyme, such hydration can by using solution washing, then with one or more organic solvents, as used easily mixed water-soluble the organic solvent such as trimethyl carbinol and Immiscible to implement in the solvent such as MTBE washing of water.

Especially, method of the present invention can make oligopeptides ester i) be coupled to oligopeptides nucleophilic reagent ii) on, and do not need based on other coupling partner excessive wherein a kind of coupling partner in a large number, with in the relatively short time with the peptide synthesized by acceptable productive rate acquisition.Oligopeptides ester i) coupling is to oligopeptides nucleophilic reagent ii) mol ratio be usually selected from the scope of 2:1 to 1:4, especially in the scope of 1:1 to 1:3, preferably in the scope of 1:1 to 1:2, the more preferably scope of 1:1 to 1:1.5, the also scope of preferred 1:1 to 1:1.2.

In preferred embodiments, linked reaction is carried out when there is not salt.This salt usually by add alkali with in and the dichloromethane solution of example as 1-2.5 volume % trifluoroacetic acid (optionally C-terminal is protected) protected oligopeptides nucleophilic reagent ii that protected oligopeptides is formed after acid excision from solid phase) salt formed.Can conveniently by protected oligopeptides from solid phase excision after with buck extraction dichloromethane layer avoid salt at (optionally C is protected good) protected oligopeptides amine nucleophilic reagent ii) existence.

In theory, the pH (once there is pH in selected reaction medium) used can select in wide limit, as long as can demonstrated enough activity by enzyme under the pH that selects.Such pH is normally known for the enzyme that will use, and based on its known hydrolytic activity in aqueous, or can may be measured routinely, makes it possible to use known substrate to enzyme under known reaction conditions.It is especially selected at about neutrality.If expected, can depend on that enzyme is to use alkalescence or acidic conditions.If expected, acid and/or alkali can be used to regulate pH, or pH can cushion with the combination of suitable bronsted lowry acids and bases bronsted lowry.Suitable bronsted lowry acids and bases bronsted lowry especially dissolves in those bronsted lowry acids and bases bronsted lowries in reaction medium, such as, be selected from ammonia and the acid being dissolvable in water organic solvent, such as acetic acid and formic acid.

In theory, the temperature used is strict, if by the temperature selected the enzyme that uses demonstrate enough activity and stability.Such temperature is normally known for the enzyme that will use, or can, by conventional determining, make it possible to use known substrate to enzyme under known reaction conditions.Usually, temperature can be at least 0 DEG C, especially at least 15 DEG C or at least 25 DEG C.Especially, if use one or more enzymes stemming from thermophilic organisms, temperature can be preferably at least 35 DEG C.Required maximum temperature depends on enzyme.Typically, such maximum temperature is well known in the art, such as shown in product data sheet when commercially available enzyme, or can by measuring routinely based on common practise.Temperature is generally less than 70 DEG C, especially less than 60 DEG C, or less than 50 DEG C.But, if especially use one or more enzymes from thermophilic organisms, higher temperature can be selected, such as, up to 90 DEG C.

For specific enzyme, those skilled in the art easily can determine best temperature condition based on common practise by normal experiment.Such as, for subtilisin, especially subtilopeptidase A (such as ), temperature can advantageously be in the scope of 25-60 DEG C.

In the method according to the invention will the oligopeptides ester i of coupling) and oligopeptides nucleophilic reagent ii) synthesize preferably by solid-phase synthesis.

PEPC terminal amino group formyl methyl (Cam) and Cam-Xxx-NH ₂ester (wherein Xxx represents the protected or not protected Argine Monohydrochloride of any side chain or non-protein amino acid) has been used in the early time in the peptide symthesis of proteases catalyze and (has such as seen Miyazawa et al.Protein & Peptide Letters; 2008; 15; 1050); and usually than alkyl ester more quickly by enzymatic condensation, but also more tend to be hydrolyzed many.

Solid phase synthesis technique has been described (such as to be seen for the synthesis of side chain completely not protected oligopeptides C-terminal Cam-ester et al.Bioorg.Med.Chem., 1998,6,891).These PEPC ends Cam-ester containing Rink or Pal connector (see schematically show 1, H.Rink, Tetrahedron letters, 1987,28,3787; F.Albericio et al.J.Org.Chem., 1990,55,3730) solid phase is synthesized and accompanies by TFA (the such as TFA/H using high density ₂o, 95/5, v/v) and cut away while side chain deprotection occurs simultaneously.Regrettably, these harshnesses cut away the unexpected partial hydrolysis also causing Cam-ester with side chain deprotection condition.

schematically show 1

The solid phase synthesis of side chain not protected PEPC end Cam-ester.

But contriver finds now, solid phase is synthesized the protected PEPC end of side chain Cam and Cam-Xxx-NH ₂ester is possible.Special Sieber or Ramage amide group connector (see schematically represent 2, Sieber, Tetrahedron letters, 1987,28,2107; Ramage et al.Tetrahedron letters, 1993,34,6599) used, peptide is (such as CH under very mild acidic conditions ₂cl ₂in 2.5 volume %TFA) cut, leave Side chain protective group and be not affected.The connector of these types is never used to the synthesis of PEPC end Cam ester up to now.

schematically show 2

Advantageously, side chain protected PEPC end Cam-ester is not come by any unexpected hydrolysis of Cam-ester from solid phase excision.Except using Sieber or Ramage connector to synthesize Cam-ester on resin, contriver have been found that now comprise 2-chlorine trityl chloride or SASRIN connector (see schematically represent 3, Barlos, Tetrahedron Letters, 1973,95,1328; Mergler et al.Tetrahedron Letters; 1988; 68,239) it is possible for solid phase being synthesized side chain protected PEPC end Cam-Xxx-OH ester (wherein Xxx represents the protected or not protected Argine Monohydrochloride of any side chain or non-protein amino acid).Unexpectedly, these Cam-Xxx-OH esters show in enzymatic fragment condensation reaction and Cam-Xxx-NH ₂ester is equally good.

schematically show 3

2-chlorine trityl chloride and SASRIN connector

Demonstrate in the literature and advantageously solid state chemistry peptide symthesis technology has been combined with enzymatic fragment condensation.But, these examples be based on the completely not protected peptide of side chain solid phase synthesis and subsequently the enzymatic condensation of these peptide fragment in water or partially aqueous solution (such as see et al.J Pept Res., 2000,55,325).

By contrast, in a preferred method of the invention, the protected peptide fragment of side chain is synthesized by solid phase technique and the enzymatic condensation subsequently in anhydrous organic solvent, and this was never disclosed before being.

Therefore, the invention still further relates to for the synthesis of comprising by general formula C (=O)-O-CX as above ₂-C (=O) N-R ₁r ₂the oligopeptides ester i of the ester moiety that (i.e. Cam-ester) represents) method.

The invention still further relates to the method for the synthesis of peptide, pass through

A) as described previously by solid phase synthesis; under mildly acidic conditions; connector is used to prepare oligopeptides ester i) between solid phase and oligopeptides; it is suitable for allowing oligopeptides ester to excise from solid phase; and retain any Side chain protective group be present on oligopeptides; and wherein oligopeptides ester excises from solid phase

B) as described previously by solid phase synthesis; under mildly acidic conditions; connector is used to prepare oligopeptides nucleophilic reagent ii) between solid phase and oligopeptides; it is suitable for allowing oligopeptides nucleophilic reagent to excise out from solid phase; and retain any Side chain protective group be present on oligopeptides; and wherein oligopeptides nucleophilic reagent excises from solid phase, and

C) subsequently by oligopeptides ester i) and oligopeptides nucleophilic reagent ii) coupling, this is coupled at and comprises in organic solvent or ORGANIC SOLVENT MIXTURES that the total amount mainly betiding liquid wherein relative to linked reaction is 0.1 below volume %, carry out under the existence of subtilisin, and be wherein removed by the water of enzyme r e lease in coupling reaction process.

Unexpectedly, enzymatic fragment condensation strategy and the most frequently used Solid phase peptide synthesis Side chain protective group based on Fmoc compatibility.Even containing large " bulky " Side chain protective group, the peptide of such as Trt, Pbf or Pmc also can by enzyme identification and with good to fabulous productive rate condensation.The protecting group of these types is never used in chemo-enzymatic peptide synthesis up to now.Be most commonly used to Fmoc/ ^tthe Side chain protective group of Bu base Solid phase peptide synthesis is: for Asp, Glu, Thr, Ser and Tyr ^tbu, for the Boc of Lys and Trp, for the Trt of His, Asn, Gln and Cys, and for Pmc or Pbf of Arg.

For Boc/Bzl base Solid phase peptide synthesis the most frequently used Side chain protective group be: for Tos or Mts of Arg; for Bzl or Cy of Asp, Glu, Thr and Ser; for the Acm of Cys; for Bom or Dnp of His; for the 2-Cl-Cbz of Lys; for the For of Trp, and for the 2-Br-Cbz of Tyr.

The present invention will be explained by following examples, but be not limited to these embodiments.

Embodiment

Unless otherwise stated, chemical obtains from commercial source, and without being further purified when using.Sieber (or Xanthenyl connector) resin and 2-chlorine trityl resin are bought from GL Biochen (China).Savinase, Esperase and Everlase buy from Novi's letter.From Bacillus (three different varient) and from the proteolytic enzyme of Bacillus licheniformis buy from Sigma.Alcalase-immozyme buys from Chiralvision.1H and 13C NMR composes record on Bruker Avance 300MHz NMR spectrograph, chemical shift with relative to TMS (0.00ppm), DMSO-d6 (for 1H be 2.50ppm or for 13C for 39.9ppm) or CDCl ₃(be 77.0ppm for 13C) provides with ppm (δ).Thin-layer chromatography (TLC) is in the upper operation of the silica gel 60F254 plate (Merck) of precoating; UV light or triketohydrindene hydrate is used to manifest spot. molecular sieve (8-12 order, Acros) 200 DEG C, decompression under activation.The trimethyl carbinol (tBuOH) is stored in these molecular sieves.Before use, tBuOH is preheated to liquid (45 DEG C).Use silica gel Merck level 9385 implement column chromatography.Analyze HPLC in HP1090 liquid chromatography, use reversed-phase column (Inertsil ODS-3, C18,5 μm, 150 × 4.6mm), carry out at 40 DEG C.UV detects at 220nm, uses the linear spectrometer of UV-VIS 204 to implement.Gradient program is: the linear gradient slope of the eluant B of 0-25min from 5% to 98%, from 25.1-30min with 5% eluant B (eluant A: 0.5mL/L methanesulfonic (MSA) water, eluant B: 0.5mL/L MSA in acetonitrile).The flow velocity of 0-25.1min is 1mL/min, 25.2-29.8min is 2mL/min, then returns 1mL/min, until stop at 30min.Sampling volume is 20 μ L.Use following Gradient program to carry out the analysis of large protected hydrophobic peptide (>10 amino acid): the linear gradient gradient of the eluant B of 0-60min from 0% to 100%; at the 60-65min 100% eluant B (water/acetonitrile solution (80/20 of eluant A:1mL/L TFA; v/v%); acetonitrile/2-the propanol/water (50/45/5, v/v/v%) of eluant B:1mL/L TFA).Use with for analyzing the identical damping fluid of HPLC and Gradient program is analyzed to implement LC-MS.Positive-ion electrospray (ESI) is used on Deca XP ion trap LC-MS (ThermoFisher Scientific), to record color atlas with the pattern of full scan (scope of 300-2000amu).The productive rate of condensation reaction is measured by the total area at comparative product peak and the total area of starting raw material, and supposes that the optical extinction coefficient of product equals the optical extinction coefficient of acry radical donor.Stationary phase post (Pursuit XRs, C18,10 μm of particle sizes, 500 × 41.4mm) is used to implement preparative HPLC in Varian PrepStar system.Alcalase-CLEA-OM buys from CLEA-Technologies, and containing 3.5wt% water; Apparent activity is that 650AGE unit often (under 40 DEG C and pH7.5, can form 1 μm of ol N-ethanoyl-Padil with 1AGE unit catalyzing N-ethanoyl-ethyl aminoacetate).This Alcalase-CLEA-OM is handled as follows before the use: 1g Alcalase-CLEA-OM is suspended in 20mL ^tin BuOH, and pulverize with spatula.After filtration, repeat this process with 20mL MTBE.Final sizing enzyme (d=0.250mm), to remove large enzyme granulate.Liquid A lcalase is handled as follows before the use: use 20mL ^tbuOH dilutes 10mL Alcalase (brown liquid solution, Novozymes type 2.5L DX), then stirs, subsequently centrifugal sediment (3500rpm), supernatant decanted liquid.By little agglomerate Eddy diffusion at 30mL ^tin BuOH, then stir, centrifugal, decant (3500rpm).The little agglomerate produced is used for according in method of the present invention.Alcalase-imibond and Alcalase-epobond buys from SPRIN technology company (Trieste, Italy), and with phosphate buffered saline buffer (10mL/g, 100mM, pH 7.5,3 times), ^tbuOH (3 times) and MTBE cleaning.Same cleaning process is applied to Alcalase-immozyme.Subtilopeptidase A and Proteinase K lyophilized powder (Sigma) are before the use by following hydration process: be dissolved in 10mL phosphate buffered saline buffer (100mM, pH7.5) by 1g lyophilized powder, then add 20mL ^tbuOH, then stirs, then centrifugation (3.500rpm), supernatant decanted liquid.By little agglomerate Eddy diffusion at 30mL ^tin BuOH, then stir, centrifugal and decant (3500rpm).This process repeats twice.

The description of the karl Fischer titration of the per-cent of the water in assaying reaction mixture will be used for.

Use Metrohm titrino 701KF, automatically measure water-content with Hydranal 2 (Sigma) karl Fischer titer reagent.Reaction mixture is filtered under an inert atmosphere, and 1.000g liquid sample is used for karl Fischer titration.Gargle with Hydranal 2 and wash dropper, and titration parameters is arranged as follows:

Extraction time=9999 seconds

Stopping criterion=skew

Be parked in skew=15 (μ L/min)

Stopping=40.0mL

Maximum rate=1.0mL/min

Minimum volume increment=min (μ L)

·I(pol.)＝10μA

Terminal=75mV

Fill rate=20.0mL/min

100mL Hydranal solvent (Sigma) is introduced in dry titration vessel, and stirs with magnetic bar.With in Hydranal 2 and Hydranal solvent, until wear rate keeps constant along with the time.1.000g sample is introduced in titration vessel, and with Hydranal 2 titration sample.Measure the amount of Hydranal 2 solution consumed, as shown in Figure 4,

X-axis: titrant consumption amount;

Y-axis: time scale titration.

The amount of consumed standard volumetric solution is inferred from consumption/time curve:

Extend titration line b to the point of crossing with x-axis

Measure from the point of crossing of X-axis to the vertical line distance of Y-axis, as the measuring vol (1cm in X-axis is equivalent to 1mL titrating solution) of the amount of the Hydranal 2 consumed.

Use the content (% by weight) of the water in following general formula calculation sample:

(v*T _t)/(10*a)

Wherein, Tt=is at t DEG C, and mg water equals 1.00mL Hydranal 2.

oligopeptides-OCam ester (oligopeptides ester i)) synthesis

Following scheme is used to carry out synthetic oligopeptide-OCam ester:

With methylene dichloride (10mL; 2x 2min) and 1-Methyl-2-Pyrrolidone (NMP; 10mL; 2x 2min) clean 1g Sieber resin (xanthenyl connector; the carrying capacity of 0.5mmol/g), and with piperidines/NMP (10mL, 1/4; v/v, 2x 8min) Fmoc-go protection.With NMP (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and NMP (10mL, 2x 2min) cleaning after, at methylene dichloride (10mL, 45min), by dicyclohexylcarbodiimide (DCC) (4 equivalent), iodoacetic acid (4 equivalent) is coupled on resin.With NMP (10mL; 2x 2min), methylene dichloride (10mL; 2x 2min) and THF (10mL; 2x 2min) cleaning after; at 50 DEG C, the amino acid (with suitable protecting group in side chain functionalities) that resin-carried Fmoc-is protected, use 4 equivalents Fmoc-Xxx-OH (wherein Xxx represents amino acid) and at DMF/THF (10mL; 1/4, v/v) in the DiPEA 20h of 10 equivalents.With DMF (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and NMP (10mL, 2x 2min) cleaning after, then standard SPPS scheme is implemented (see Fmoc Solid Phase Peptide Synthesis by W.C.Chan and P.D.White, Oxford university press, 2004) to extend peptide.15min is implemented from the resin excision 2.5 volume % trifluoroacetic acids (TFA) be used in methylene dichloride (10mL every gram resin).Clean resin with methylene dichloride, the filtrate of merging is concentrated into 1/3 of original volume in a vacuum.Subsequently, add isopropanol/water (1/3, v/v), mixture is concentrated into 1/2 of original volume in a vacuum.Oligopeptides-O-Cam the ester of precipitation is filtered out, and with water cleaning twice, then from acetonitrile/water (3/1, v/v) freeze-drying.Analyze according to HPLC, product is usually with the productive rate of >90%, and the purity of >95% obtains.

fmoc-Ala-O-CH ₂ the synthesis of-COOH

1mmol Fmoc-Ala-OH is dissolved in the anhydrous THF/DMF of 100mL (8/2, v/v), then adds the 2-iodo acetic acid tertiary butyl ester of 2 equivalents and the DiPEA of 2.5 equivalents.This mixture is shaken 20 hours with 150rpm at 50 DEG C.Then, removing volatiles under vacuum, and resistates is dissolved in 250mLEtOAc and 250mL NaHCO again ₃in the mixture of saturated aqueous solution.Be separated two-phase, use NaHCO ₃saturated aqueous solution (250mL, 1x), the HCl aqueous solution (250mL, pH 1,2x), salt solution (250mL, 1x) wash organic layer, use Na ₂sO ₄drying, concentrates in a vacuum, with toluene (50mL, 2x) and CHCl ₃(50mL, 2x) coevaporation volatile matter.Then, 10mL TFA/H is added ₂(95/5, v/v, stir the mixture O 1h, then adds 100mL IPA/H ₂o (1/3, v/v).Remove 50mL volatile matter in a vacuum, throw out is filtered and uses 50mL H ₂o washs (2x).Remaining thick ester, by preparative HPLC purifying, analyzes the productive rate of acquisition 52% and the purity of > 98% according to HPLC.

fmoc-Gln (Trt)-O-CH ₂ the synthesis of-COOH

1mmol Fmoc-Gln (Trt)-OH is dissolved in the anhydrous THF/DMF of 100mL (8/2, v/v), then adds the 2-iodo acetic acid benzene methyl of 2 equivalents and the DiPEA of 2.5 equivalents.This mixture is vibrated 20 hours with 150rpm at 50 DEG C.Then, removing volatiles under vacuum, and resistates is dissolved in 250mLEtOAc and 250mL NaHCO again ₃in saturated aqueous solution.Be separated two-phase, use NaHCO ₃saturated aqueous solution (250mL, 1x), the HCl aqueous solution (250mL, pH 1,2x), salt solution (250mL, 1x) washs organic layer, uses Na ₂sO ₄drying, concentrates in a vacuum, with toluene (50mL, 2x) and CHCl ₃(50mL, 2x) coevaporation volatile matter.Residue is dissolved in 250mL MeOH/ toluene (1/1, v/v), then uses the Pd/C of 1g 10%, at 25 DEG C, 5bar H ₂lower hydrogenolysis spends the night.After solids removed by filtration, removing volatiles under vacuum.The thick ester produced, by preparative HPLC purifying, analyzes the productive rate of acquisition 63% and the purity of > 98% according to HPLC.

oligopeptides-Ocam-Xxx-NH ₂ ester is (with R ₂ oligopeptides ester i), R ₂ for having C-terminal carboxylic acid amides the amino-acid residue of functional group) synthesis

1g Sieber resin (xanthenyl connector, the carrying capacity of 0.5mmol/g) is cleaned with methylene dichloride (10mL, 2x 2min), NMP 10mL (2x 2min); and with piperidines/NMP (10mL; 1/4, v/v, 2x 8min) Fmoc-go protection.With NMP (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and NMP (10mL, 2x 2min) cleaning after, be used in NMP (10mL, Fmoc-Xxx-OH (4 equivalent) is coupled on resin by the HBTU (4 equivalent) 45min), HOBt (4 equivalent) and DiPEA (8 equivalent).With NMP (10mL; 2x 2min), methylene dichloride (10mL; 2x 2min) and NMP (10mL; 2x 2min) cleaning after; use piperidines/NMP (1/4; v/v; 2x 8min) amino acid Fmoc-is gone protection; resin NMP (10mL; 2x 2min), methylene dichloride (10mL, 2x2min) and NMP (10mL, 2x 2min) cleaning after; be used in the HBTU (2 equivalent) in NMP (10mL, 90min), HOBt (2 equivalent) and DiPEA (4 equivalent) coupling Fmoc-Xxx-O-CH ₂-COOH (2 equivalent).With NMP (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and NMP (10mL, 2x 2min) cleaning after, execution standard SPPS scheme is (see Fmoc Solid Phase Peptide Synthesis by W.C.Chan and P.D.White, Oxford university press, 2004) to extend peptide.Oligopeptides-Ocam-Xxx-NH ₂the cracking of ester is identical with scheme 2 with purifying.Analyze according to HPLC, product is usually with the productive rate of >90%, and the purity of >95% obtains.

oligopeptides-Ocam-Xxx-OH ester is (with R ₂ oligopeptides ester i), R ₂ for with C-terminal carboxylic-acid functional group amino-acid residue) synthesis

With methylene dichloride (10mL, 2x 2min) clean 1g Trityl resin (the chloro-chlorotrityl connector of 2-, the carrying capacity of 1.0mmol/g), Fmoc-Xxx-OH (2 equivalent) is coupled on resin by the DiPEA (5 equivalent) be used in methylene dichloride (10mL, 30min).After DMF (10mL, 2x 2min) cleaning, unreacted chlorotrityl methylene dichloride/MeOH/DiPEA (10mL, 80/15/5, v/v/v, 2x 10min) blocks a shot.With NMP (10mL; 2x 2min), methylene dichloride (10mL; 2x 2min) and NMP (10mL; 2x 2min) cleaning after, use piperidines/NMP (10ml, 1/4; v/v; 2x 8min) amino acid Fmoc-is gone protection, be then used in DCC (4 equivalent) the coupling iodoacetic acid (4 equivalent) in methylene dichloride (10mL, 45min).With NMP (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and THF (10mL, 2x 2min) cleaning after, at 50 DEG C, with the DiPEA process resin 20h of Fmoc-Xxx-OH (4 equivalent) and 10 equivalents in DMF/THF (10mL, 1/4, v/v).With DMF (10mL, 2x 2min), methylene dichloride (10mL, 2x 2min) and NMP (10mL, 2x 2min) cleaning after, execution standard SPPS scheme is (see Fmoc Solid Phase Peptide Synthesis by W.C.Chan and P.D.White, Oxford university press, 2004) to extend peptide.The cracking of oligopeptides-Ocam-Xxx-OH ester is identical with scheme 2 with purifying.Analyze according to HPLC, product is usually with the productive rate of >90%, and the purity of >95% obtains.

oligopeptides C-terminal acid amides nucleophilic reagent (oligopeptides nucleophilic reagent ii)) synthesis

Use standard SPPS scheme (Fmoc Solid Phase Peptide Synthesis by W.C.Chan and P.D.White, Oxford university press, 2004) at the upper synthetic oligopeptide C-terminal acid amides nucleophilic reagent of Sieber resin (xanthenyl connector, the carrying capacity of 0.5mmol/g).The 2.5 volume % trifluoroacetic acids (TFA) be used in methylene dichloride (10mL every gram resin) from the excision resin implement 15min.With 10mL methylene dichloride cleaning resin twice, the filtrate of merging uses 10mL NaHCO ₃saturated aqueous solution washes twice, and is then concentrated into 1/3 of its original volume in a vacuum.Precipitation, washs identical with scheme 2 with drying.Analyze according to HPLC, product is usually with the productive rate of >90%, and the purity of >95% obtains.

embodiment 1

protected oligopeptides Acibenzolar i) with protected oligopeptides C-terminal acid amides nucleophilic reagent ii) fragment coupling

3 μm of ol protected oligopeptides Acibenzolars and 4.5 μm of ol protected oligopeptides C-terminal acid amides nucleophilic reagents are dissolved in 0.5mL methylene dichloride, and add the molecular sieve of 10mg pulverizing.Then, the mother liquor (being kept in molecular sieve) containing Alcalase-CLEA-OM (20mg/mL in methylene dichloride) is added.This mixture, at 37 DEG C, with 200rpm vibration 24h, and is analyzed with LC-MS.

Table 1

^aafter reaction 7

Table 1 indicate multiple protected oligopeptides C-terminal ester can with the coupling of multiple protected oligopeptides C-terminal acid amides nucleophilic reagent, produce the peptide prod of nearly at least 19 amino acid lengths of high yield.In addition, any fragment used or not in the condensation reaction of a large amount of stoichiometric excess, and the productive rate obtained.

embodiment 2

in multi-solvents, Ac-Asp (OtBu)-Leu-Ser (tBu)-Lys (Boc)-Gln-Ocam (R1 and R2 oligopeptides i for hydrogen)) and H-Met-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Val-NH ₂ (oligopeptides ii) fragment coupling

By 2.2mmol Ac-Asp (OtBu)-Leu-Ser (tBu)-Lys (Boc)-Gln-OCam (Seq.ID No:16) and 3.3mmol H-Met-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Val-NH ₂(Seq.ID No:21) is dissolved in 100 μ L DMF.Then, molecular sieve, 10mg Alcalase-CLEA-OM and 900 μ L solvents that 10mg pulverizes are added.This mixture, at 37 DEG C, with 200rpm vibration 24h, and is analyzed with HPLC.

^ahFIP; ^b2,2,2 tfifluoroethyl alcohol; ^cuse 50mg Alcalase-CLEA-OM.

This table indicates all kinds of SOLVENTS can be used to protected oligopeptide fragments linked reaction.

embodiment 3

relatively Ocam-Xxx-NH ₂ ester is (with R ₂ oligopeptides ester i), R ₂ for with C-terminal carboxylic acid amides official the amino-acid residue that can roll into a ball)

By various Cbz-Val-Ala-OCam-Xxx-NH ₂ester and Cbz-Val-Ala-OCam are with H-Phe-NH ₂linked reaction compare.By 0.028mmol Cbz-Val-Ala-OCam-Xxx-NH ₂or Cbz-Val-Ala-OCam is added in 1.5mLTHF (containing 10 volume %DMF), H-Phe-NH containing 5 molecular sieves ₂in (1.5 equivalent).Reaction mixture is stirred 30min at 50 DEG C, then adds the Alcalase of 50mg solid on diatomite (decalite).Stir 1h at 50 DEG C after, the reaction mixture of 100 μ L parts is added in 900 μ L DMF, and uses HPLC analytic sample.From Cbz-Val-Ala-Phe-NH ₂productive rate is determined in product calibration chart.It should be noted, after 1h, the difference between various oligopeptides Acibenzolar becomes clearly.The reaction times that use extends and/or more multienzyme, all reactions can transform completely.

This table shows multiple C-terminal-Ocam-Xxx-NH ₂ester can be used to enzymatic linked reaction.

embodiment 4

ocam ester and Ocam-Leu-NH ₂ the comparison of ester

By H-Met-Glu (tBu)-Glu (tBu)-Glu (the tBu)-Ala-NH of 3 μm of ol protected oligopeptides C-terminal esters and 4.5 μm of ol ₂(Seq.ID No:21) is dissolved in 0.5mL methylene dichloride, and adds the molecular sieve of 10mg pulverizing.Then, the 0.5mL storing solution (being kept in molecular sieve) containing Alcalase-CLEA-OM (in methylene dichloride, 40mg/mL) is added.By this mixture at 37 DEG C, shake 48 hours with 200rpm, and analyzed by LC-MS.

The results show that the result for [2+1] coupling of display in embodiment 3 can be converted into peptide fragment coupling, and at least in some cases, compared to unsubstituted-OCam-ester, the C-terminal-OCam-Xxx-NH of protected oligopeptides ₂the use of ester is favourable for coupling rate and productive rate.

embodiment 5

oligopeptides-OCam-Xxx-NH ₂ ester (C-terminal carboxylic acid amides functional group) and oligopeptides-OCam-Xxx-OH ester the comparison of (C-terminal carboxylic acid functional)

As described in Example 3, by Cbz-Val-Ala-OCam-Leu-NH ₂with Cbz-Val-Ala-OCam-Leu-OH and H-Phe-NH ₂enzymatic condensation.By Cbz-Val-Gln (Trt)-OCam-Gly-NH ₂with Cbz-Val-Gln (Trt)-OCam-Gly-OH and H-Phe-NH as described in Example 3 ₂carry out enzymatic condensation, but use the enzyme of 5 times amount.Before enzymatic coupling, use in piperidines (1 equivalent) and carboxylic moiety.

This result shows C-end-Ocam-Xxx-NH ₂ester and-Ocam-Xxx-OH ester may be used for enzymatic linked reaction, and show suitable speed of reaction at least in some cases.

embodiment 6

multiple enzyme and use in fixed form

By 2.2mmol Ac-Asp (OtBu)-Leu-Ser (tBu)-Lys (Boc)-Gln-OCam and 3.3mmol H-Met-Glu (OtBu)-Glu (OtBu)-Glu (OtBu)-Val-NH ₂be dissolved in 100 μ L DMF.Then, molecular sieve, 10mg enzyme and 900 μ L MTBE that 10mg pulverizes are added.By these mixtures at 37 DEG C, with 200rpm vibration 24h, and analyze with HPLC.Following enzyme creates good productive rate (> 80%): Alcalase-CLEA-OM, Alcalase-imibond, Alcalase-epobond, Alcalase-immozyme, Liquid Alcalase, subtilopeptidase A, Proteinase K.

By 2.2mmol Cbz-Phe-OCam and 3.3mmol H-Phe-NH ₂be dissolved in 100 μ L DMF.Then, molecular sieve, 10mg enzyme and 900 μ L MTBE that 10mg pulverizes are added.By these mixtures at 37 DEG C, with 200rpm vibration 24h, and analyze with HPLC.Following enzyme creates good productive rate (> 80%): Savinase, Esperase, Everlase, from the proteolytic enzyme (three kinds available varient) of Bacillus, the proteolytic enzyme from Bacillus licheniformis (Bacillus licheniformis).

embodiment 7

protected oligopeptides activation Cam ester i) and H-Leu-Phe-NH ₂ fragment condensation

3 μm of protected oligopeptides acry radical donors of ol are dissolved in 0.5mL methylene dichloride, and add the molecular sieve of 10mg pulverizing.Then, the H-Leu-Phe-NH containing 12 μm of ol/mL in methylene dichloride is added ₂with the 0.5mL mother liquor (being kept in molecular sieve) of the Alcalase-CLEA-OM of 20mg/mL.By this mixture at 37 DEG C, with 200rpm vibration 24h, and analyzed by LC-MS.After it should be noted that 24h, the difference between multiple Cam ester becomes clearly.The reaction times that use extends and/or more multienzyme, institute responds to become and transforms completely.

As observed from entry 1-4, multiple Side chain protective group may be used for the C-terminal GIn residue of oligopeptides C-terminal ester, but if this C-terminal GIn residue has not protected side chain functionalities, is favourable.The protected oligopeptides C-terminal ester that entry 5-13 demonstrates with multiple amino acids sequence and Side chain protective group may be used for enzymatic linked reaction.

embodiment 8

protected oligopeptides C-terminal acid amides nucleophilic reagent ii) with the coupling of Cbz-Phe-OCam

3 μm of ol protected oligopeptides C-terminal acid amides nucleophilic reagents are dissolved in 0.5mL methylene dichloride, and add the molecular sieve of 10mg pulverizing.Then, the storing solution (being kept in molecular sieve) of the 0.5mL of the Alcalase-CLEA-OM of Cbz-Phe-OCam and 20mg/mL containing 12 μm of ol/mL in methylene dichloride is added.By this mixture at 37 DEG C, with 200rpm vibration 24h, and analyzed by LC-MS.After it should be noted that 24h, the difference between various protected oligopeptides C-terminal acid amides nucleophilic reagent becomes clearly.The reaction times that use extends and/or more multienzyme, institute responds to become and transforms completely.

The protected oligopeptides C-terminal acid amides nucleophilic reagent that entry 1-9 demonstrates with multiple amino acids sequence and Side chain protective group may be used for enzymatic linked reaction.

embodiment 9

water-content is in the impact on synthesis/hydrolysing rate

By 2.2 μm of ol Cbz-Phe-OCam and 4.4 μm ol H-Phe-NH ₂be dissolved in 1mL methylene dichloride, and add 5mg Alcalase-CLEA-OM (prepare six parts identical like this mixture).0,0.1,0.5,1.0,2.0 and 3.0 μ L water (being equivalent to the water of ≈ 0,0.01,0.05,0.1,0.2 and 0.3 volume % in the reactive mixture) are added in these mixtures.By these six parts of mixtures at 37 DEG C, with 200rpm vibration 2h, and analyze with HPLC.

S/H ratio is defined as formed dipeptides product C bz-Phe-Phe-NH ₂amount (mmol) divided by the amount (mmol) of formed hydrolysate Cbz-Phe (mmol).As from comprise seen in fig. 1, low water concentration is most important for avoiding Cam ester hydrolysis.

embodiment 10

by the enzyme reactivate of rehydration

Inactivation

By 5mg Alcalase-CLEA-OM and 25mg molecular sieve vibrates respectively in 1mL DMF, 1mL methylene dichloride and 1mL DMF/MTBE (1/9, v/v) at 37 DEG C.For three kinds of solvents, prepare three parts of identical like this mixtures.For all three kinds of solvents, after 1,24 and 48h, filter three parts of mixtures respectively, add the H-Phe-NH containing 5mg Cbz-Phe-OCam and 1.5 equivalents in each case ₂1mLDMF/THF (1/9, v/v), and at 37 DEG C oscillatory reaction mixture.In order to compare, 1mL is contained the H-Phe-NH of 5mg Cbz-Phe-OCam and 1.5 equivalents ₂1mL DMF/THF (1/9, v/v) and 5mg Alcalase-CLEA-OM and 25mg not vibrate in organic solvent before molecular sieve vibrates at 37 DEG C.Take out sample after 1h, and change into Cbz-Phe-Phe-NH by HPLC mensuration ₂transformation efficiency.By the Cbz-Phe-Phe-NH will obtained with the Alcalase-CLEA-OM of organic solvent process ₂amount (mmol) divided by the Cbz-Phe-Phe-NH obtained with undressed Alcalase-CLEA-OM ₂amount (mmol) x 100% calculate relative reactivity.

As visible in appended accompanying drawing 2, Alcalase-CLEA-OM is containing slow inactivation in the organic solvent of the drying of molecular sieve.

Reactivate

By 5mg Alcalase-CLEA-OM and 25mg molecular sieve vibrates respectively in 1mL DMF, 1mL methylene dichloride and 1mL DMF/MTBE (1/9, v/v) at 37 DEG C.For all three kinds of solvents, prepare three parts of identical mixtures.For all three kinds of solvents, three parts of mixtures are filtered respectively after 1,24 and 48h, add the 1mL (1/1 containing 5mg Cbz-Val-Phe-OMe in each case, v/v) DMF/ phosphate buffer soln (100mM, pH=7.5), oscillatory reaction mixture and at 37 DEG C.In order to compare, 1mL is contained (1/1, v/v) DMF/ phosphate buffer soln (100mM, the pH=7.5) of 5mg Cbz-Val-Phe-Ome and 5mg Alcalase-CLEA-OM and 25mg do not vibrated in organic solvent before molecular sieve vibrates at 37 DEG C.Take out sample after 1h, and measured the transformation efficiency changing into Cbz-Val-Phe-OH by HPLC.By the amount (mmol) of the Cbz-Val-Phe-OH obtained by hydrolysis with the Alcalase-CLEA-OM of organic solvent process is calculated relative reactivity divided by with undressed Alcalase-CLEA-OM by amount (mmol) x 100% being hydrolyzed the Cbz-Val-Phe-OH obtained.

As visible in appended accompanying drawing 3, containing the Alcalase-CLEA-OM of slow inactivation in the organic solvent of the drying of molecular sieve, can in aqueous buffer solutions reactivate.

embodiment 11

use the molecular sieve of multiple content enzyme deactivation and in aqueous buffer solutions reactivate

Inactivation

10,20,30 and 50mg molecular sieve is added respectively in four parts of the 10mg Alcalase-CLEA-OM in 0.5mL methylene dichloride same mixtures by mixture at 37 DEG C, vibrate 20 hours with 200rpm.Then, add containing 0.2mM Cbz-Phe-OCam and 0.3mM H-Phe-NH ₂0.5mL methylene dichloride, and by reaction mixture at 37 DEG C, to vibrate with 200rpm.Take out sample after 1h, and change into Cbz-Phe-Phe-NH by HPLC mensuration ₂transformation efficiency.By the Cbz-Phe-Phe-NH will obtained by enzymatic linked reaction ₂the Cbz-Phe-Phe-NH that obtains divided by the enzymatic linked reaction (i.e. the situation of 10mg molecular sieve) by having the highest transformation efficiency of amount (mmol) ₂amount (mmol) x 100% calculate relative reactivity.

Using makes the Alcalase-CLEA-OM of its inactivation synthesize Cbz-Phe-Phe-NH with the molecular sieve of multiple consumption ₂.

As visible in above form, higher enzyme deactivation can be caused owing to using the condition of relatively large molecular sieve and too dry.

Reactivate

Nitrogen gas stream is used to be evaporated by the methylene dichloride of above four parts of reaction mixtures.Then, add the 1mL 100mM phosphate buffered saline buffer (pH8) containing 5mg Cbz-Asn-OMe, reaction mixture is vibrated with 200rpm at 37 DEG C.Take out sample after 1h, and measured the transformation efficiency changing into Cbz-Asn-OH by HPLC.Relative reactivity is calculated by amount (mmol) x 100% of the Cbz-Asn-OH amount (mmol) of the Cbz-Asn-OH obtained by enzymically hydrolyse obtained divided by the enzymically hydrolyse (i.e. the situation of 50mg molecular sieve) by having the highest transformation efficiency.

Use and make the Alcalase-CLEA-OM of its inactivation be hydrolyzed Cbz-Asn-OMe with the molecular sieve of multiple consumption.

As from above form, the activity of water-disintegrable Alcalase-CLEA-OM is suitable with the level of deactivation before it, and the level of deactivation before not relying on it, indicating enzymic activity can recover in aqueous.

What use in sequence table writes a Chinese character in simplified form:

Ac=ethanoyl

Boc=tertbutyloxycarbonyl

NH2=amine

OCam=carboxylic acid amides methyl esters

OtBu=tert-butyl ester

Pbf=2,2,4,6,7-pentamethyl-dihydrobenzo-furans-5-alkylsulfonyl

TBu=tertiary butyl

Tmob=2,4,6-trimethoxy benzyl

Trt=trityl

Xan=xanthenyl

accompanying drawing:

Fig. 1: water-content is on the impact of synthesis/hydrolysis (S/H) ratio of Alcalase-CLEA-OM.

Fig. 2: Alcalase-CLEA-OM is containing inactivation in the organic solvent (mixture) that three kinds of molecular sieve are different.

Fig. 3: Alcalase-CLEA-OM inactivation in aqueous buffer solutions.

Fig. 4: the amount of Hydranal 2 solution consumed in time.

Claims (17)

1. the method for enzyme' s catalysis oligopeptides, described method comprise by

I) the oligopeptides ester of 4 or more amino-acid residues is comprised,

-it comprises at least two amino-acid residues, the side chain functionalities that each amino-acid residue is protected with protected base, and comprises the C-terminal ester of activation, and described C-terminal ester is by general formula C (=O)-O-CX ₂-C (=O) N-R ₁r ₂represent, wherein, each X represents hydrogen atom or alkyl or aryl independently of one another, and R ₁represent hydrogen atom or alkyl or aryl, R ₂represent hydrogen atom or alkyl or aryl or with the amino-acid residue of C-terminal carboxylic acid amides or carboxylic acid functional or peptide residue; described amino-acid residue or peptide residue are optionally in the side chain functionalities or protected in one or more side chain functionalities of peptide residue of amino-acid residue

-and wherein, oligopeptides ester optionally comprises N-terminal protection,

With

Ii) the oligopeptides nucleophilic reagent of 4 or more amino-acid residues is comprised,

-it comprises N-terminal amido, and at least two amino-acid residues, the side chain functionalities that each amino-acid residue is protected with protected base, and

-wherein, described oligopeptides nucleophilic reagent optionally comprises C-terminal protection,

Coupling, described be coupled to comprise mainly betide in 0.1 volume % of total amount of liquid wherein or the organic solvent of following water or ORGANIC SOLVENT MIXTURES relative to linked reaction, implement under the existence of subtilisin, and wherein remove the water by enzyme r e lease during coupling reaction process.

2. method according to claim 1, is characterized in that, oligopeptides ester obtains by using the solid phase synthesis of connector.

3. method according to claim 2, is characterized in that, connector is Sieber or Ramage connector.

4. according to the method in claim 1-3 described in any one, it is characterized in that, in all side chain functionalities at least 50% is protected.

5. according to the method in claim 1-4 described in any one, it is characterized in that, each X represents hydrogen atom.

6. according to the method in claim 1-5 described in any one, it is characterized in that, R ₁and R ₂both represent hydrogen atom.

7. according to the method in claim 1-6 described in any one, it is characterized in that, R ₁represent hydrogen atom, R ₂represent the amino-acid residue with C-terminal carboxylic acid amides or carboxylic acid functional or peptide residue, optionally, the side chain functionalities of its amino-acid residue or one or more side chain functionalities of peptide residue protected.

8. according to the method in claim 1-7 described in any one, it is characterized in that, except the side chain functionalities of the C-terminal amino-acid residue of oligopeptides ester, all side chain functionalities of oligopeptides ester and oligopeptides nucleophilic reagent are protected.

9. according to the method in claim 1-8 described in any one, it is characterized in that, subtilisin is the varient of wild-type subtilisin.

10. according to the method in claim 1-9 described in any one, it is characterized in that, use subtilisin in fixed form.

11., according to the method in claim 1-10 described in any one, is characterized in that, subtilisin is fixed with the form of cross-linked enzyme aggregate.

12. according to the method in claim 1-11 described in any one, it is characterized in that, organic solvent or ORGANIC SOLVENT MIXTURES comprise MTBE, THF, Me-THF, 1,2-glycol dimethyl ether, methylene dichloride, 1,2-ethylene dichloride, TFE, DMF, NMP, DMA or DMSO.

13. methods according to claim 12, is characterized in that, organic solvent or ORGANIC SOLVENT MIXTURES comprise the mixture of the mixture of MTBE, MTBE and DMF or NMP or DMA or DMSO, methylene dichloride or methylene dichloride and DMF or NMP or DMA or DMSO.

14., according to the method in claim 1-13 described in any one, is characterized in that, organic solvent or ORGANIC SOLVENT MIXTURES have 0.05 volume % or following water-content.

15., according to the method in claim 1-14 described in any one, is characterized in that, removing is by the water of enzyme r e lease continuously.

16., according to the method in claim 1-15 described in any one, is characterized in that, use molecular sieve removing by the water of enzyme r e lease.

17., according to the method in claim 1-16 described in any one, is characterized in that, linked reaction is carried out under the condition that there is not salt.